(a) Field of the Invention
This invention relates to a method of detecting genetic variation in individuals by PCR amplification of the locus of interest, transferring the resulting PCR products to a membrane filter, hybridizing with allele-specific-oligonucleotides (ASOs), and visualizing the results. Such a method is in the field of diagnostics, pharmacogenetics, cancer therapeutics, and drug metabolism. In particular, the present invention relates to the detection of genetic polymorphisms in gene encoding xenobiotics metabolizing enzymes CYP1A1, CYP2D6, CYP3A4, and NAT2.
(b) Description of Prior Art
It is recognized that marked interindividual differences exist in enzymes that metabolize xenobiotics and that this variability can be genetically determined. Considerable progress has been made in identifying a number of individual enzymes that are subject to genetic polymorphism. A polymorphism is generally considered to be a stably inherited trait. Genetic variation means differences between individuals in regard to the base sequence of their DNA. Genotyping involves identification of (defined) genetic mutations that, in a particular case, give rise to the specific drug metabolism phenotypes. These mutations include genetic alterations that lead to increased activity, absence of an active protein (null allele) or production of a mutant protein with altered catalytic capacity. A number of types of genetic polymorphism in xenobiotics metabolizing enzymes are relevant to clinical practice. These include those relating to N-ace-tyltransferase 2 (NAT2) and the cytochromes P450 1A1 (CYP1A1), 3A4 (CYP3A4) and 2D6 (CYP2D6). Genetic variation can be detected by means of electrophoresis of DNA fragments, generated by digestion of PCR products with a restriction enzyme the so-called PCR-RFLP approach. This method that has proven useful in screening for genetic mutations associated with altered metabolism of drugs and/or cancer susceptibility. It consists of amplification of a specific region of the gene of interest by PCR followed by digestion of the amplified DNA product with restriction endonucleases. Restriction endonucleases have the capacity to digest DNA with a high degree of nucleotide sequence specificity. Thus, point mutations within the recognition sequence of a specific restriction endonuclease may be detected through determining whether the DNA of interest serves as a substrate for that endonuclease. These studies are routinely carried out by comparing the size of digestion products generated from a DNA substrate amplified from control subject DNA with respect to study subject DNAS. The size of the digestion products is easily evaluated by agarose gel electrophoresis with ethidium bromide staining and UV transillumination. In this case the position of the bands varies between individuals, as a result of gain or loss of the recognition site for the restriction enzyme used, this is restriction fragment length polymorphism (RFLP). However, the PCR-RFLP is not well suited for genotyping a considerable number of samples that are usually analyzed in such investigations.
It would be highly desirable to be provided with a PCR-based assay to detect point mutations in CYP1A1, CYP2D6, CYP3A4 and NAT2.